Gadolinium expressed lipid nanoparticles for magnetic resonance imaging

ABSTRACT

Lipid nanoparticles expressing metal ions and methods for using the compositions for magnetic resonance imaging.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2010/024324, filed Feb. 16, 2010, which application claims thebenefit of U.S. Provisional Application No. 61/152,459, filed Feb. 13,2009, and U.S. Provisional Application No. 61/162,989, filed Mar. 24,2009. Each application is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Contract No. AI52663 awarded by the National Institutes for Health/National Instituteof Allergy and Infectious Diseases. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Early diagnosis of lymph node involvement is essential to determinetreatment for most types of cancer. Lymphadenectomy and histologicalevaluation is currently the gold standard, but is not ideal because itis invasive and can produce false negatives. Magnetic resonance (MR)imaging has become a powerful and non-invasive tool for detecting thespread of cancer to the lymph nodes. Standard MR imaging relies on sizeand morphology criteria to determine occult lymphoid tissues includinglymph node metastasis, which has as low as 60% accuracy. MR contrastenhancing agents are becoming more widely used due to their usefulnessin early tumor detection. Contrast enhancing agents diffuse intometastatic lymph nodes and healthy lymph nodes at different ratescausing “filling defects.” Predicting lymph node metastasis usingfilling defects from contrast agents as opposed to size criteria canincrease the sensitivity from 29% to 93%.

Generally, there are two types of MR contrast enhancing agents.Superparamagnetic contrast agents have a low r2/r1 ratio and create darkspots in T2- and T2*-weighted images. These are usually based on ironoxide particles and are referred to as negative contrast agents. On theother hand, paramagnetic contrast agents increase the r1 relaxivity andhave a high r2/r1 ratio, creating bright spots in T1 weighted MR images.These contrast agents are known as positive contrast agents and areusually complexes of gadolinium (Gd³⁺).

T1 shortening comes about through dipole-dipole interactions with theprotons in water so Gd³⁺, with its seven unpaired electrons, is theoptimal choice for a T1 relaxation agent. Because Gd³⁺ is a heavy metaltoxin, it is commonly delivered as a tightly bound linear or macroscopicchelate. Chelated forms of Gd³⁺ reduce toxicity by preventing cellularuptake of free Gd³⁺ and by limiting the clearance almost exclusively torenal filtration that resulted in renal toxicities. Despite reducingtoxicity, the rapid clearance and small molecular size of gadoliniumchelates mean that low levels of Gd³⁺ accumulates in the lymph nodes,making these agents a poor choice for MR lymphography. In addition, theFDA posted a warning about the risk of serious nephrogenic systemicfibrosis for all commercially available gadolinium contrast agent toidentify well-perfused tissues and organs in subjects with acute orchronic renal insufficiency.

Liposomes and lipid nanoparticles containing Gd³⁺ have severaladvantages for MR contrast imaging of lymph nodes. Liposomes as well aslipid nanoparticles can lower the toxicity by encapsulating or bindingto their surfaces a large amount of Gd³⁺. However, the rapid clearancemechanism of intravenously (IV) administered liposomes does notsignificantly improve liposome-associated Gd³⁺ accumulation in the lymphnodes. Only a fraction of the lipid nanoparticles in blood arephagocytosed by reticuloendothelial cells, and only a fraction of thosecells traverse to lymphatic system. Thus, IV administered liposomesprovide indirect targeting of the lymphatic system and lymph nodes.However, this approach results in a majority of Gd³⁺ carrying liposomeseliminated through reticuloendothelial cells in blood. Thepharmacokinetics of liposomes in blood can be optimized for lymph nodeaccumulation through size and surface modification. Reducing thediameter of the liposomes below 200 nm decreases phagocytic dependentclearance and increases the circulation time in the blood. Addingpolyethylene glycol (PEG, commonly referred to as PEGylation) to theliposome surface can also increase the circulation time and stability ofliposomes.

Currently, gadolinium chelated with diethylenentriaminepentaacetyl(DTPA) provides contrast in magnetic resonance imaging to identifypathogenic tissues. Unfortunately, the soluble Gd-DTPA complexesapproved for clinical use, such as OMNISCAN, are cleared within a fewminutes and do not provide sufficient concentrations or time in lymphoidtissues. In addition, the residual fraction of gadolinium can lead tofibrosis in patients with renal insufficiency.

Despite the advances in the development of contrast agents, a needexists for improved contrast agents having longer in vivo life, providesufficient concentration in tissues to be analyzed, and low residualgadolinium concentrations to avoid side effects. The present inventionseeks to fulfill this need and provide further related advantages.

SUMMARY OF THE INVENTION

The present inventions provides compositions expressing metal ions andmethods for using the compositions.

In one aspect, the invention provides a composition, comprising a lipid,a polyalkylene-containing lipid, and a lipid-containing metal chelator.

Representative lipids include phospholipids, sphingolipids, cholesteroland steroid derivatives, bile acids and derivatives, cardilipin,acyl-glycerides and derivatives, glycolipids, acyl-peptides, fattyacids, carbohydrate-based polymers, functionalized silica, polyanhydridepolymers, polylactate-glycolate polymers, and bioploymers. In oneembodiment, the lipid is a phospholipid. Representative phospholipidsinclude 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); dipalmitoylphosphatidylcholine;

dimysristoyl phosphatidyl choline; dioleoyl phosphatidyl choline;trans-esterified phospholipids derived from eggs, soybean, flaxseed, andthe like; and phosphatidylcholine substituted with phosphatidylethanolamine, phosphatidylglycerol, phosphatidyl serine, andphosphatidic acids.

Representative polyalkylene-containing lipids includepolyoxyethylene-containing lipids or polyoxypropylene-containing lipids.In one embodiment, the polyalkylene-containing lipid is a phospholipidfunctionalized with polyethylene glycol such asN-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanol-amine(mPEG-2000-DSPE).

Representative lipid-containing metal chelators include1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetyl(DSPE-DTPA), tetraazacyclododecane, tetraacety(gadodiamide)-PE, andlipid-functionalized-[N,N-bis[2-[bis(carboxymethyl)amino]-ethyl]-glycinato-(5″)].In one embodiment, the lipid-containing metal chelator comprises aPEGylated lipid moiety. In one embodiment, the lipid-containing metalchelator is a PEGylated DTPA. In other embodiments, the lipid-containingmetal chelator is a DSPE-BOPTA, a DSPE-DO3A, and a DSPE-DOTA.

In one embodiment, the composition further includes a targeting moiety.Representative targeting moieties include proteins, polypeptides,peptides, antibodies or fragments thereof, small molecules, sugars orpolysaccharides or derivatives thereof, and nucleic acids.

In one embodiment, the composition of the invention has the form of ananoparticle. In one embodiment, the nanoparticle has a diameter of fromabout 5 nm to about 2 μm. In one embodiment, the nanoparticle has adiameter of from about 50 nm to about 100 μm.

The composition of the invention can further include a metal ion.Suitable metal ions include paramagnetic metal ions and ions ofradio-isotopes. Representative paramagnetic metal ions include Gd³⁺,Cu²⁺, Fe³⁺, Fe²⁺, and Mn²⁺ ions. Representative ions of radio-isotopesinclude ⁶⁸Ga, ⁵⁵Co, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, and ¹¹¹In ions.

In another aspect, the invention provides administrable compositionsincluding a carrier and a plurality of the nanoparticles of theinvention. Suitable carriers include pharmaceutically acceptablecarriers such as saline for injection or dextrose for injection.

In further aspect, the invention provides a method for imaging oftissues, comprising administering to a subject to be imaged an effectiveamount of the composition of the invention. Representative tissues thatcan be imaged by the method include lymphoid, cardiovascular, liver,kidney, brain, heart, muscle, and gastrointestinal tract tissues, andother tissues accessible by the lymphatic or vascular (blood) systems.

In another aspect, the invention provides a method for delivering aradio-cancer therapeutic agent to a cancer cell, comprisingadministering to a cancer cell an effective amount of the composition ofthe invention that includes anion of a radio-isotope.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIG. 1 is a graph illustrating reduction of calcein fluorescence as afunction of Gd³⁺: DTPA-PE (m/m) ratio for a representative formulationof the invention. The formulation was incubated with calcein and itsfluorescence measured. Free Gd³⁺ binds to calcein and reduces itsfluorescence.

FIGS. 2A and 2B compare relaxivity as a function of Gd³⁺ concentration.FIG. 2A compares Gd³⁺ dose effect on T1 relaxation rate; R₁ (1/T1)values of Gd³⁺ concentrations (μmol/mL) measured in a 1.5T magneticresonance (MR) scanner using a standard spin-echo sequence for arepresentative formulation of the invention (Gd-DPTA-PE: mPEG-PE: DSPC)compared to other formulations. FIG. 2B compares Gd³⁺ dose effect on T2relaxation rate; R₂ (1/T2) values of Gd³⁺ concentrations (μmol/mL)measured in a 1.5T magnetic resonance (MR) scanner using a standardspin-echo sequence for a representative formulation of the invention(Gd-DPTA-PE: mPEG-PE: DSPC) compared to other formulations.

FIG. 3 illustrates time-course coronal images of ventral cavity of M.Nemestrina up to 24 hours after injection of a representativeformulation of the invention (Gd-DPTA-PE: mPEG-PE: DSPC). FIGS. 3A-3Dare time sequence images of the liver before (3A) and after 20 min (3B),6 hr (3C), and 24 hr (3D) administration. The top panel illustrates timesequence images of lymph nodes. The lymph nodes and liver show highcontrast compared to background tissues.

FIG. 4A is a graph comparing MR dynamic contrast enhanced imageintensity of the lymph nodes (LN 1, LN 2, and LN3) and liver (Liver) inM. Nemestrina up to 24 hr after subcutaneous injection of 24.4 μmol/kgof a representative formulation of the invention (Gd-DPTA-PE: mPEG-PE:DSPC). Results are for images shown in FIGS. 3A-3D. FIG. 4B is a graphcomparing the time course of tissue specific MR signal (lymph node)compared to adjacent control tissue.

FIGS. 5A-5D are time course MR images of a rat after intravenousinjection of a representative formulation of the invention (0.01 mmol/kgGd-DPTA-PE: mPEG-PE:

DSPC): pre-dose (5A); 5 min (5B); 14 min (5C); and 24 hr (5D). Contrastenhancement is localized mainly in vasculature and vascularized tissuesin lymph nodes at 5 min and 14 min. The arrow in 5C indicates thebeginning of gadolinium elimination into the gastrointestinal tractthrough the biliary route. By 24 hr (5D) most of the contrast agent iseliminated and the residual fraction appears in the intestine.

FIGS. 6A-6C are time course MR images of a rat after intravenousinjection of a commercially available gadolinium-based contrast agent(0.05 mmol/kg Gd-DPTA, OMNISCAN): pre-dose (6A); 5 min (6B); and 15 min(6C). Contrast enhancement is diffused throughout and does not localizein the vasculature, well-perfused tissues, or the lymph nodes at either5 min or 15 min. Distribution of contrast media to periphery andextremities is apparent.

FIGS. 7A-7D compare time course MR images of a rat after intravenousinjection of a commercially available gadolinium-based contrast agent(0.05 mmol/kg Gd-DPTA, OMNISCAN, pre-dose (7A), 5 min (7B), and 15 min(7C)) an MR image of a rat 15 min after administration of arepresentative formulation of the invention (0.01 mmol/kg Gd-DPTA-PE:mPEG-PE: DSPC).

FIGS. 8A-8F compare dose response MR images of a rat after intravenousinjection of a representative formulation of the invention (Gd-DPTA-PE:mPEG-PE: DSPC): 0.0 mmol/kg (8A); 0.00125 mmol/kg (8B); 0.0025 mmol/kg(8C); 0.005 mmol/kg (8D); and 0.010 mmol/kg (8E and 8F). Lymph nodes areclearly apparent in FIG. 8F.

FIGS. 9A-9C compare MR images of rats after intravenous injection of twocommercially available gadolinium-based contrast agents (0.05 mmol/kgMS-325, 9A; 0.05 mmol/kg MAGNEVIST Gd-DPTA, 9B) and a representativeformulation of the invention (0.01 mmol/kg Gd-DPTA-PE: mPEG-PE: DSPC,9C). Images were acquired near peak enhancement, about 1 to 2 min afteradministration.

FIG. 10 is a schematic illustration of the preparation of arepresentative lipid-containing gadolinium chelate: DSPE-DOTA-Gd.

FIG. 11 illustrates three metal chelators useful for preparingrepresentative lipid-containing chelates: p-SCN-Bn-DOTA; CHX-A″-DTPA;and p-SCN-Bn-DTPA.

FIG. 12 is a schematic illustration of the preparation of representativelipid-containing gadolinium chelates.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions provides compositions expressing metal ions andmethods for using the compositions. In one embodiment, the compositionsare lipid nanoparticles that include paramagnetic metal ions and areuseful for magnetic resonance imaging. In another embodiment, thecompositions are lipid nanoparticles that include ions of radio-isotopesand are useful for delivery of radio-cancer therapeutic agents.

In one aspect, the invention provides compositions and methods formagnetic resonance imaging. The compositions and methods enhancegadolinium distribution and accumulation in lymphatics. The inventionprovides a gadolinium composition (referred to herein as “Gd-DTPA-lipidnanoparticle”) that is suitable for both intravenous and subcutaneousadministration. Subcutaneous administration allows direct access tolymphatic system. The composition enhances T1 weighted MR signal in thelymph nodes as well as increases the residence time of the contrastagent in the lymphatics. Upon intravenous administration, thecomposition exhibits at least 100-fold enhancement over soluble Gd-DTPAas a vascular imaging agent and eliminates predominantly throughbiliary, rather than renal clearance. The composition was shown tosignificantly increase signal-to-noise ratio by more than 300-fold forMR visualization of lymph nodes in macaques.

The composition of the invention includes a lipid, apolyalkylene-containing lipid, and a lipid-containing metal chelator. Inone embodiment, the composition further includes a chelated metal ion.

In one embodiment, the composition of the invention is a chelator- (ormetal chelate-) expressing particle. As used herein, the term“expressing” refers to the particle presenting or having available thechelator or chelated metal for activity. As noted above, in oneembodiment, the composition of the invention is a lipid nanoparticlehaving chelated gadolinium ion (Gd⁺³) (e.g., Gd-DTPA-lipidnanoparticle). In the lipid nanoparticle, chelated gadolinium ion isexpressed.

The lipid nanoparticles of the invention are biocompatible and arereadily administered. The nanoparticles have a diameter of from about 5nm to about 2 μm. In one embodiment, the nanoparticles have a diameterof from about 10 nm to about 100 μm. In one embodiment, thenanoparticles have a diameter of about 70 nm.

As noted above, the composition of the invention (e.g., lipidnanoparticles) includes a lipid, a polyalkylene-containing lipid, and alipid-containing metal chelator.

Lipids. The lipid component of the nanoparticles of the inventioncomprise the nanoparticle core.

Representative lipids useful in the compositions include phospholipids,sphingolipids, cholesterol and steroid derivatives, bile acids andderivatives, cardilipin, acyl-glycerides and derivatives, glycolipids,acyl-peptides, fatty acids, carbohydrate-based polymers (e.g., cellulosepolymers), suitably functionalized silica, lipophilic polymers (e.g.,polyanhydrides, polylactate-glycolate), and lipophilic bioploymers(e.g., proteins, sugar polymers).

In one embodiment, the lipid is disteroylamidomethylamine.

In one embodiment, the lipid is a phospholipid. Representativephospholipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);dipalmitoyl phosphatidylcholine; dimysristoyl phosphatidyl choline;dioleoyl phosphatidyl choline; trans-esterified phospholipids derivedfrom eggs, soybean, flaxseed, and the like; and phosphatidylcholinesubstituted with phosphatidyl ethanolamine, phosphatidylglycerol,phosphatidyl serine, and phosphatidic acids. In one embodiment, thephospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

Polyalkylene-containing lipids. The polyalkylene-containing lipidcomponent of the nanoparticles of the invention serve as surfacehydrating agents.

Representative polyalkylene-containing lipids includepolyoxyethylene-containing lipids and polyoxypropylene-containinglipids. In one embodiment, the polyalkylene-containing lipid is aphospholipid functionalized with polyethylene glycol (e.g., PEGylatedphospholipid). Suitable PEGylated phospholipids include a polyethyleneglycol having a number average molecular weight of from about 500 toabout 20,000. In one embodiment, the PEGylated phospholipid isN-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine(mPEG-2000-DSPE) (also referred to herein as “mPEG-DSPE” and “mPEG-PE”).

In addition to polyalkylene-containing lipid, in other embodiments, thesurface hydrating agent is hydrophilic biomaterials such as acarbohydrate polymer, a polyamine, a polyvinyl pyrrolidone, apoly(aspartate), or a poly(L-amino acid).

Other useful surface hydrating agents include covalent conjugates ofpolyethoxyl, polymethylene glycol, or propylene glycol and a lipid orother hydrophobic moiety (e.g., long chain hydrocarbon).

The surface hydrating agent is preferably present from about 5 to about50 mole percent of the composition (i.e., lipid, polyalkylene-containinglipid (surface hydrating agent), and lipid-containing metal chelator).

Lipid-containing metal chelator. The lipid-containing metal chelatorcomponent of the nanoparticles of the invention are expressed on thesurface of the nanoparticle and serve to chelate metal ions. Suitablelipid-containing metal chelators include two moieties: (1) a lipidmoiety and (2) a metal chelator moiety.

Representative lipid-containing metal chelators include1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetyl(DSPE-DTPA), tetraazacyclododecane, tetraacety(gadodiamide orOMNISCAN)-PE, and lipid-functionalized-[N,N-bis[24bis(carboxymethyl)amino]ethyl]-glycinato-(5″)] (MAGNEVIST).

Representative metal chelators include BOPTA, DO3A, and DOTA chelators.

In one embodiment, the metal chelator includes a PEGylated lipid moiety.Representative PEGylated metal chelators include DSPE-BOPTA, aDSPE-DO3A, and a DSPE-DOTA. In one embodiment, the metal chelator is aPEGylated DTPA (DPTA-PE).

The metal chelator is preferably present from about 5 to about 50 molepercent of the lipid, polyalkylene-containing lipid (surface hydratingagent), and metal chelator.

Chelated metal ion. The compositions of the invention are effectivecarriers of metal ions. In these embodiments, the composition (e.g.,lipid nanoparticles) further includes a chelated metal ion.

For MR applications, useful metal ions include paramagnetic metal ions.Representative paramagnetic metal ions include Gd³⁺, Cu²⁺, Fe³⁺, Fe²⁺,and Mn²⁺ ions.

For other applications such as imaging and therapeutic ion delivery,useful metal ions include ions of radio-isotopes. Representativeradio-isotopes include ions of ⁶⁸Ga, ⁵⁵CO, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, and ¹¹¹In.

For embodiments that include chelated metal ions, the ratio of metalion: metal chelator is 0.1-1.0:1.0 (less than or equal to 1:1).

Targeting agents. The compositions of the invention can be used totarget specific tissues. In these embodiments, the composition (e.g.,lipid nanoparticles) further includes a targeting moiety. Representativetargeting moieties include proteins, polypeptide, and peptides;antibodies and derivatives (fragments); small molecules; sugars,polysaccharides, and derivatives; and nucleic acids, such as nucleotidepolymers (e.g., aptamers), DNAs; and RNAs. Representative targetingmoiety targets include cancer cells and virus infected cells.

Lipid nanoparticle formulations. The lipid nanoparticles of theinvention can be formulated into compositions for administration.Suitable compositions for administration include a carrier and aplurality of the lipid nanoparticles. Representative carriers includepharmaceutically acceptable carriers, such as saline for injection ordextrose for injection.

The lipid nanoparticle of the invention is not a liposome and does notform liposomes when formulated.

Methods for tissue imaging. In another aspect, the invention providesmethods for imaging tissues (e.g., occluded tissues). In one embodiment,the method includes administering to a subject to be imaged adiagnostically effective amount of a composition of the invention. Thecomposition can be administered by a variety of techniques includingsubcutaneously and intravenously. The method is effective for imagingtissues such as lymphoid, cardiovascular, liver, kidney, brain, heart,muscle, and gastrointestinal tract tissues, and other tissues accessibleby the lymphatic or vascular (blood) systems. The method is effectivefor imaging the tissues above to determine whether the tissues areoccluded. For magnetic resonance imaging methods, the compositionincludes a paramagnetic metal ion (e.g., Gd³⁺).

In general, the effective amount is from about 0.001 to about 5 mmolmetal/kg subject. In one embodiment, the effective amount is from about0.005 to about 0.050 mmol metal/kg subject. In one embodiment, theeffective amount is about 0.010 mmol metal/kg subject.

Methods for radio-cancer therapeutic agent delivery. In another aspect,the invention provides methods for delivering a radio-cancer therapeuticagent to a cancer cell. In one embodiment, the method includesadministering to a subject in need thereof a therapeutically effectiveamount of a composition of the invention in which the chelated metal ionis a radio-isotope (e.g., ⁶⁸Ga, ⁵⁵Co, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, and ¹¹¹In). Thecomposition can be administered by a variety of techniques includingsubcutaneously and intravenously. The method is effective for deliveryto tissues such as lymphoid, cardiovascular, liver, kidney, brain,heart, muscle, and gastrointestinal tract tissues, and other tissuesaccessible by the lymphatic or vascular (blood) systems.

The following is a description of the preparation, characterization, andimaging results for representative lipid nanoparticles of the invention.

Lipid nanoparticles were prepared composed of 10 mole percent ofsurface-bound DTPA. These lipid nanoparticles containeddistearoyl-phosphatidylcholine and PEGylated lipid, mPEG-2000-DSPE. Theywere allowed to complex with Gd³⁺ (presented as Gd³⁺—Cl⁻) at varyingGd³⁺-to-DTPA-PE mole ratios. The presence of free Gd³⁺ in the admixturewas determined by the ability of free Gd³⁺ to quench the fluorescence ofcalcein. With up to a Gd³⁺-to-DTPA-PE mole ratio of 4, no free Gd³⁺could be detected by the calcein quenching assay. At a 6 or higherGd³⁺-to-DTPA-PE mole ratio, free or unbound Gd³⁺ was detected (see FIG.1). To determine the effects of Gd³⁺ on DTPA-expressing lipidnanoparticles, we determined the particle diameter by photon correlationspectroscopy. The diameter of DTPA-expressing lipid nanoparticles wasGd³⁺ concentration dependent. At or below a Gd³⁺-to-DTPA mole ratio of1, the presence of Gd³⁺ did not influence the diameter of lipidnanoparticles. At a Gd³⁺-to-DTPA mole ratio of 2, the apparent diameterof Gd³⁺-DTPA lipid nanoparticle increases by about 2- to 3-fold, whilethere is no apparent decrease in the degree of Gd associated withDTPA-lipid nanoparticles (see Table 1). These lipid nanoparticlesappeared to be stable as no significant change in diameter was detectedover 24 hr at room temperature. Collectively, these data suggest thatthe ratio of Gd³⁺-to-DTPA influence the degree of Gd³⁺ incorporationinto Gd-DTPA lipid nanoparticles and their apparent diameters. At orbelow Gd³⁺-to-DTPA mole ratio of 2, substantially all Gd³⁺ wasassociated with lipid nanoparticles. In one embodiment, the Gd³⁺-to-DTPAmole ratio in the composition of the invention is about 1.

The contrast properties of the Gd³⁺-expressed lipid nanoparticles wasdetermined by comparing the effects of the various Gd³⁺ formulations onthe R1 (1/T1) relaxivity of Gd³⁺. Lipid nanoparticles composed ofdistearoyl-phosphatidylcholine (DSPC) with or without PEGylated lipid(mPEG-2000-DSPE, referred to herein as “mPEG-DSPE” or “mPEG-PE”) andfixed Gd³⁺-to-DTPA mole ratio at 1. The T1 and T2 measurements werecollected with a 1.5 T MR scanner. A clinically-used Gd-DTPA preparation(OMNISCAN, commercially available from GE Healthcare, Princeton, N.J.)was included as a comparison. As shown in FIG. 2A, the representativecomposition of the invention, Gd-DTPA lipid nanoparticle containingmPEG-PE (Gd-DTPA-PE: mPEG-PE: DSPC), exhibited significantly higher R1value than other preparations, including OMNISCAN and lipidnanoparticles that did not contain mPEG. The effects of mPEG on Gd-DTPAlipid nanoparticles are less for T2 measurement (see FIG. 2B). However,the R1 and R2 values for both Gd-DTPA lipid nanoparticle compositions(Gd-DTPA-PE: mPEG-PE: DSPC and Gd-DTPA-PE: DSPC) were significantlyhigher than that of the soluble Gd-DTPA commercial preparation(OMNISCAN) or free Gd³⁺ in solution (Gd w/o DTPA). To our knowledge theR1 values for Gd-DTPA nanoparticles of the invention are the highestvalue observed to date including those reported with Gd^(3±)-expressedliposome containing PEG. In fact these results were more than 10-foldhigher than the data collected with Gd-DTPA liposomes containing egglectin and cholesterol that express PEG (MW=5000) linked to eggtransesterified PE, instead of mPEG linked to DSPE.

Both of the nanoparticle formulations containing Gd³⁺ had higher R1values than soluble Gd-DTPA. As expected, the DSPC and DSPC plusmPEG-2000-PE control formulations without Gd³⁺ showed no significanteffect on relaxivity. The data indicates that the PEG-containingGd-DTPA-lipid nanoparticles provide a much greater increase in R1compared to the other formulations. Up to a 100-fold increase in R1relaxivity was achieved when compared to the commercially availableOMNISCAN. The Gd-DTPA nanoparticle formulation without surface PEG(Gd-DTPA-PE: DSPC) also showed higher R1 values than OMNISCAN, but muchless than the PEG-containing Gd-DTPA-lipid nanoparticle formulation(Gd-DTPA-PE: mPEG-PE: DSPC) (see FIG. 2A).

Because positive contrast generated by Gd³⁺ in an MR image is dependanton a low R2/R1 ratio, changes of R2 values were also determined. FIG. 2Bdemonstrates the effects of the various Gd³⁺ formulations on the R2(1/T2) relaxivity measurements. Once again the PEGylated Gd-DTPA-lipidnanoparticles (Gd-DTPA-PE: mPEG-PE: DSPC and Gd-DTPA-PE: DSPC) showed agreater increase in R2 compared to the other formulas, however themagnitude of the change was less. The mPEGylated Gd-DTPA-lipidnanoparticles (Gd-DTPA-PE: mPEG-PE: DSPC) showed about eight-foldincrease in R2, compared to OMNISCAN. The Gd-DTPA in DSPC (without mPEG)nanoparticle formulation (Gd-DTPA-PE: DSPC) showed a greater increase inR2 than R1, which may limit its effectiveness as a positive contrastagent formulation. The DSPC and DSPC plus mPEG-2000-PE controlformulations without Gd³⁺ (DTPA-PE: mPEG-PE: DSPC and DTPA-PE: DSPC)again showed no effect on relaxivity. The relaxivity experiments showthat the mPEGylated Gd-DTPA-lipid nanoparticle formulation greatlyincreases the R1 relaxivity compared to other formulations. However, athigh concentrations (greater than 3 μmol/ml), the T1 spillover into T2becomes significant, leading to an apparent reduction of T1 signal (datanot shown). Optimal T1 tissue contrast could be obtained with minimumGd³⁺ concentration. These data clearly demonstrate much higher contrast“potency” than that provided by the commercial product, OMNISCAN.

The use of the Gd-DTPA lipid nanoparticles containing mPEG-2000-PE(Gd-DTPA-PE: mPEG-PE: DSPC or “Gd-DTPA lipid nanoparticles”) for MRimaging studies in primates is described below.

FIGS. 3A-3D shows several coronal MR images of ventral cavity of M.Nemestrina both pre-contrast and up to 24 hours following subcutaneousinjection of Gd³⁺-DTPA-lipid nanoparticles. The auxiliary lymph nodesare clearly visible with a high degree of intensity enhancement comparedto the surrounding tissue (FIG. 3 top panel). The enhancement of thelymph nodes can be seen as early as twenty minutes while lasting up totwenty four hours. FIG. 4 shows the time course of dynamic contrastenhanced MRI, showing relative intensities of various organs vs. time.This data shows that the Gd³⁺-DTPA-lipid nanoparticles quickly reach thelymph nodes tissue within twenty minutes after injection, and maintainthe contrast enhancement for at least twenty four hours. Compared to alow signal that dissipates within 20 min in lymphatic system of animalstreated intravenously with soluble Gd-DTPA (OMNISCAN preparations), asingle subcutaneous Gd-DTPA lipid nanoparticles dose provided greatlyenhanced signal for extended time.

The MR image enhancing property of the Gd-DTPA-lipid nanoparticles canbe used to minimize the IV dose need to produce vascular imageenhancement and also reduce renal burden. Administration of 0.01mmole/kg Gd-DTPA nanoparticles (about ⅕ of current dose for human) inrats produced a high quality MR image with clearly discernable centraland peripheral vasculature of rat within 5 min (FIG. 5B). It begins toclear through the bile and gut within 15 min (FIG. 5C), and by theclearance process appeared to complete by 24 hr (FIG. 5D). In contrast,0.05 mmole soluble Gd-DTPA (OMNISCAN) produce diffuse contrastlocalization with no vascular definition; it also appeared to distributeto periphery an extremities (FIG. 6A). In a limited dose-response study,only about to 10-20-fold lower dose of the Gd-DTPA-lipid nanoparticles,compared to current Gd-DTPA formulation, is needed in rat to produceequivalent or better definition of contrast enhanced MR images in rats.The 0.0025 mmole/kg Gd in DTPA-lipid nanoparticles produce equivalentimage quality of 0.05 mmole/kg Gd-DTPA (OMNISCAN) preparation.Therefore, the Gd-DTPA-lipid nanoparticles may overcome challenges inthe clinical use with currently approve Gd contrast agents due to renalinsufficiency and neurotoxicity.

The PEGylated lipid nanoparticles of the invention having surface-boundgadolinium ion exhibited a great improvement over other preparations incontrast enhanced MR lymphography and vascular imaging. These lipidnanoparticles showed high degree of accumulation in the lymph nodesafter subcutaneous injection. The contrast enhancement in lymphoidtissue begins within 20 minutes of injection and is maintained for 24hours. When given intravenously this agent produced high quality imagesof vasculature in much higher sensitivity than the current agents.Intravenously administered lipid nanoparticles are cleared almostexclusively through biliary route and appeared to complete within 24 hr.Surface modification by adding mPEG in lipid nanoparticles increased theMR signal of Gd³⁺ through coordination of water molecules. This leads toa much higher R1 relaxivity and lymph node image enhancement. The lipidnanoparticle formulation may allow using a low dose to achieve a highsignal-to-noise MR contrast ratio for increasing the metastatic nodaldiscrimination and allowing for a much wider time frame for imaging. Thepotentially lower dose and more favorable elimination route of Gd³⁺needed for MR contrast could provide higher safety margin.

The formulations of the invention provide effective contrast atrelatively low dose compared to currently available and approvedcontrast agents. FIGS. 8A-8F compare dose response MR images of a ratafter intravenous injection of a representative formulation of theinvention (Gd-DPTA-PE: mPEG-PE: DSPC): 0.0 mmol/kg (8A); 0.00125 mmol/kg(8B); 0.0025 mmol/kg (8C); 0.005 mmol/kg (8D); and 0.010 mmol/kg (8E and8F). Lymph nodes are clearly apparent in FIG. 8F.

As noted above, the formulations of the invention offer advantages overcurrently available and approved contrast agents. FIGS. 9A-9C compare MRimages of rats after intravenous injection of two commercially availablegadolinium-based contrast agents (0.05 mmol/kg MS-325, EPIXPharmaceuticals, Inc., Cambridge, Mass., FIG. 9A; and 0.05 mmol/kgMAGNEVIST Gd-DPTA, Bayer HealthCare Pharmaceuticals, FIG. 9B) and arepresentative formulation of the invention (0.01 mmol/kg Gd-DPTA-PE:mPEG-PE: DSPC, FIG. 9C). Images were acquired near peak enhancement,about 1 to 2 min after administration. As can be seen from the images,the representative formulation of the invention demonstratessignificantly greater contrast than the currently available agents.

FIG. 10 is a synthetic scheme for preparing a representative Gdcomplexes useful in the invention (DSPE-DOTA-Gd) by reacting DSPE withp-SCN-Bn-DOTA followed by Gd metallation. FIG. 11 illustrates threerepresentative chelating agents (isothiocyanates, —N═C═S or —NCS)(p-SCN-Bn-DOTA, CHX-A″-DTPA, and p-SCN-Bn-DTPA) that are reactive towardphospholipids and useful in the invention. FIG. 12 illustrates fourrepresentative lipophilic compounds (DSA, Diether PE, DSPE-PEG(2000)Amine, and DSPE) and a synthetic scheme for preparing a representativeGd complex useful in the invention (DSPE-DOTA-Gd) by reacting DSPE withp-SCN-Bn-DOTA followed by Gd metallation. Lipid-containing metalchelators useful in the invention are readily prepared as shown in FIGS.10 and 12. Suitably reactive metal chelators (e.g.,isothiocyanate-functionalized metal chelators, see FIG. 11) are reactedwith lipid compounds containing suitably reactive groups (e.g., aminogroups in DSPE, DSPE-PEG(2000) Amine, Diether PE, DSA, see FIGS. 10 and12) to provide lipid-containing metal chelators in which the lipidmoiety is covalently coupled to the metal chelator. Forisothiocyanate-functionalized metal chelators and reactiveamine-containing lipids, the product lipid-containing metal chelatorincludes a thiourea (—NH—C(═S)—NH—) linkage coupling the lipid to thechelator. It will be appreciated that other suitably reactive metalchelators (e.g., isocyanate) and lipid compounds containing suitablyreactive groups (e.g., alcohol) can provide lipid-containing metalchelators useful in the invention. Metallation of the chelators providesthe metal ion-containing compounds.

TABLE 1 Effects of the Gd³⁺ to DTPA mole ratio on particle size andstability of Gd³⁺-lipid nanoparticles^(a). Gd³⁺:DTPA Particle diameter(nm)^(c) (m/m) ratio^(b) After preparation At 24 hr Free or unboundGd^(3+d) 0 52 ± 0.05 65 ± 0.3 — 0.5 46 ± 0.09  49 ± 0.07 1.4 ± 2.7% 1 41± 0.1  48 ± 0.1 1.6 ± 4.8% 2 137 ± 0.15  145 ± 0.2  0.8 ± 2.0% ^(a)DTPAis expressed on lipid nanoparticles composed of DSPC, mPEG-PE andDTPA-PE (8:1:0.9 mole ratio, as described in Example 1. ^(b)Thenanoparticles were exposed to GdCl₃ at indicated DTPA to Gd³⁺ moleratio. ^(c)The diameter of DTPA expressed nanoparticles were measured byphoton correlation spectroscopy and data expressed were mean ± SD ofquadruplicate samples at indicated time points. ^(d)The presence ofunbound or free Gd³⁺ was estimated with calcein fluorescence quenchingassay.

The following examples are provided for the purpose of illustrating, notlimiting, the invention.

EXAMLPES Example 1 Materials and Methods

Lipid nanoparticle preparation.1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids,Ala.),N-(carbonyl-methoxypolethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phospoethanolamine(mPEG-2000-DSPE, Genzyme, Mass.), and1,2-distearoyl-sn-glycero-3-phophoethanolamine-N-DTPA (DSPE-DTPA, AvantiPolar Lipids, AL) were combined in chloroform (DSPC: mPEG-DSPE:DSPE-DTPA) in a ratio of 8:2:1 and dried into a thin film under nitrogenand then under high vacuum overnight. The mPEG-DSPE containing PEGpolymers of various molecular weights (or chain lengths) are alsopurchased from Genzyme, Mass. At this point phosphate buffered saline(PBS, pH 7.4) was added to the film and sonicated in a bath-typesonicator (Laboratory Supplies Company, New York). The vesicle diameter,as measured by dynamic light scattering using a Malvern Zetasizer 5000photon correlation spectroscopy (Malvern Instruments, PA), was 50 nm.The nanoparticles in suspension were mixed with gadolinium (III)chloride hexahydrate (Aldrich, St Louis, Mo.) for 20 minutes atindicated mole ratio to form Gd-DTPA-lipid nanoparticles. To determinethe unbound Gd³⁺, the nanoparticles were incubated with calcein (0.5 μM)(Sigma, St Louis, Mo.) in PBS, pH 7.4, and the fluorescence was measuredat 485/535 nm using a Victor3V 1420 multilabel counter (PerkinElmer,Waltham, Mass.). Free ionic Gd³⁺ quenches calcein fluorescence in [Gd³⁺]dependent manner. To determine the final Gd concentration, elemental Gdmass was determined using the inductively coupled plasma atomic emissionspectrometry. The particles along with control particles without Gd³⁺were used in the studies described herein.

Relaxivity studies. Dilutions of Gd-DTPA-lipid nanoparticles wereprepared with Gd³⁺ concentrations between 0-5 μmol/ml. For comparisonseveral samples were prepared from commercial agents such as OMNISCAN(Gd-DTPA-BMA) with Gd³⁺ concentrations from 0-5 μmol/ml. The relaxationtime T1 was measured using the standard spin-echo sequence on a 1.5T MRscanner with a volume head coil as RF receiver. For T1 measurements, TEwas fixed to 9 ms and seven TR were 133, 200, 300, 500, 750, 1000 and2000 ms, respectively. For T2 measurements, TR was fixed to 2000 ms andfour TE were 15, 30, 45, and 60 ms, respectively. The imagingintensities were fitted to obtain the corresponding T1 and T2 values,which were plotted versus Gd³⁺ concentration.

Primate lymphatic MRI Study. In vivo imaging of the lymph nodes usingGd-DTPA-lipid nanoparticles for dynamic contrast enhanced (DCE) MRI wasperformed in a 1.5T MR scanner. The pigtailed macaque (M. Nemestrina)was anesthetized with inhaled isofluorane (1-2%) and closely monitoredduring the experiments. A pre-contrast image of the primate was recordedto determine proper lymph node location and fine-tune the imagingparameters. The animal was removed from the MR scanner and injectedsubcutaneously at four sites. Each injection site received 2, 5, 5, and8.5 mL, respectively of 6.1 μmol/ml Gd-DTPA-lipid nanoparticles to allowprobing of dose effects and contrast diffusion from injection sites. Thetotal dose of Gd is estimated to be 24.4 μmol/kg for the primatestudies. The images were recorded on a Signa 1.5T Scanner using asurface coil 12×12 inch. A standard spin-echo imaging sequence was usedwith TR=500 ms, TE=15 ms, slice thickness of 3 mm, 21 slices, slicegap=0.5 mm, FOV (field of view)=320×320 mm², matrix size=512×512, whichgives an in-plane resolution of 0.63×0.63 mm² and a temporal resolutionis 3.1 min.

Rat vascular MRI study. In vivo imaging of the rat using Gd-DTPA-lipidnanoparticles for dynamic contrast enhanced (DCE) MRI was performed in a3.0T MR scanner. The rats (SD) was anesthetized with inhaled isofluorane(1-2%) and closely monitored during the experiments. A pre-contrastimage of the rat was recorded to determine proper location, orientationand fine-tune the imaging parameters. The animal was removed from the MRscanner and injected with 400 μL of indicated Gd contrast media throughfemoral vein. The images were recorded on a Signa 1.5T Scanner using asurface coil 12×12 inch. A standard spin-echo imaging sequence was usedwith TR=500 ms, TE=15 ms, slice thickness of 3 mm, 21 slices, slicegap=0.5 mm, FOV (field of view)=320×320 mm², matrix size=512×512, whichgives an in-plane resolution of 0.63×0.63 mm² and a temporal resolutionis 3.1 min.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A nanoparticle, comprising: (a) a lipid; (b) apolyalkylene-containing lipid; and (c) a lipid-containing metalchelator, wherein the nanoparticle is not a liposome.
 2. Thenanoparticle of claim 1, wherein the lipid is selected from the groupconsisting of phospholipids, sphingolipids, cholesterol and steroidderivatives, bile acids and derivatives, cardilipin, acyl-glycerides andderivatives, glycolipids, acyl-peptides, and fatty acids.
 3. (canceled)4. The nanoparticle of claim 1, wherein the lipid is a phospholipid isselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); dipalmitoylphosphatidylcholine; dimysristoyl phosphatidyl choline; dioleoylphosphatidyl choline; trans-esterified phospholipids derived from eggs,soybean, flaxseed, and the like; and phosphatidylcholine substitutedwith phosphatidyl ethanolamine, phosphatidylglycerol, phosphatidylserine, and phosphatidic acids.
 5. The nanoparticle of claim 1, whereinthe polyalkylene-containing lipid is selected from the group consistingof polyoxyethylene-containing lipids and polyoxypropylene-containinglipids.
 6. The nanoparticle of claim 1, wherein thepolyalkylene-containing lipid is a phospholipid functionalized withpolyethylene glycol.
 7. (canceled)
 8. The nanoparticle of claim 6,wherein the phospholipid functionalized with polyethylene glycol isN-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine(mPEG-2000-DSPE).
 9. The nanoparticle of claim 1, wherein thelipid-containing metal chelator is selected from the group consisting ofDSPE-BOPTA, DSPE-DO3A, DSPE-DOTA,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetyl(DSPE-DTPA), tetraazacyclododecane, tetraacety(gadodiamide)-PE, andlipid-functionalized-[N,N-bis[2-[bis(carboxymethyl)amino]-ethyl]-glycinato-(5″)].10-14. (canceled)
 15. The nanoparticle of claim 1 further comprising atargeting moiety.
 16. The nanoparticle of claim 15, wherein thetargeting moiety is selected from the group consisting of a protein, apolypeptide, a peptide, an antibody or fragment thereof, a smallmolecule, a sugar or polysaccharide or derivative thereof, and a nucleicacid. 17-20. (canceled)
 21. The nanoparticle of claim 1 furthercomprising a chelated metal ion.
 22. (canceled)
 23. The nanoparticle ofclaim 21 wherein the chelated metal ion is a paramagnetic metal ion. 24.The nanoparticle of claim 23, wherein the paramagnetic metal ion isselected from the group consisting of Gd³⁺, Cu²⁺, Fe³⁺, Fe²⁺, and Mn²⁺.25. The nanoparticle of claim 21, wherein the chelated metal ion is anion of a radio-isotope.
 26. The nanoparticle of claim 25, wherein theion of a radio-isotope is selected from the group consisting of ions of⁶⁸Ga, ⁵⁵Co, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, and ¹¹¹In.
 27. A composition, comprising acarrier and a plurality of the nanoparticles of claim
 21. 28-29.(canceled)
 30. A method for imaging of tissues, comprising administeringto a subject to be imaged an effective amount of the composition ofclaim
 23. 31. The method of claim 30, wherein the composition isadministered subcutaneously or intravenously.
 32. (canceled)
 33. Themethod of claim 30, wherein the tissue is selected from the groupconsisting of lymphoid, cardiovascular, liver, kidney, brain, heart,muscle, and gastrointestinal tract tissues, and other tissues accessibleby the lymphatic or vascular (blood) systems. 34-36. (canceled)
 37. Amethod for delivering a radio-cancer therapeutic agent to a cancer cell,comprising administering to a cancer cell an effective amount of thecomposition of claim
 25. 38. The method of claim 37, wherein thecomposition is administered subcutaneously or intravenously. 39.(canceled)